Technical Field of the Disclosure
The method and apparatus disclosed herein relates in general to the cryopreservation of biological materials. More specifically, the present disclosure relates to a method and apparatus for cryopreservation of various cell types such as hematopoietic stem and progenitor cells, mesenchymal stem and progenitor cells and endothelial progenitor cells found in normal blood, placental-cord blood, bone marrow or the stromal vascular fraction cells resident in adipose tissue.
Description of the Related Art
Hematopoietic stem and progenitor cells make up a very small percentage of the nucleated cells normally found in bone marrow, cord blood or adipose tissue and are even rarer in normal blood. For instance, only approximately one in five hundred bone marrow cells are nucleated cells, and only one in one thousand nucleated bone marrow cells is a progenitor cell; and stem cells occur at an even lower frequency.
Reconstitution of the hematopoietic system for patients in need has conventionally been accomplished by transfusion into a patient of hematopoietic stem and progenitor cells from the bone marrow or cord blood of suitable Human Leukocyte Antigen (HLA) matched donors. These donor stem and progenitor cells migrate to the interior of bones, take up residence, and then typically multiply and replace the blood cells responsible for protective immunity, tissue repair, clotting, oxygen transport and other functions of the patient's blood. In a successful transplant treatment, the blood, bone marrow, spleen, thymus, and other organs of immunity are repopulated with cells derived from the donor.
These types of transplants have been used with increasing success to treat various fatal or crippling diseases, including certain types of anemia such as aplastic anemia, immune deficiencies, cancers such as lymphoma or leukemia, carcinomas, various solid tumors, genetic disorders of hematopoiesis and inherited storage diseases. Improvements in hematopoietic reconstitution techniques are thus greatly needed.
It is known that hematopoietic reconstitution is best accomplished when the donor is a perfectly matched (genetically identical) sibling. But often a sibling donor is either not a perfect match, or is unwilling or unavailable to donate. Realistically, most often the best available match is from cord blood or bone marrow from an unrelated donor that is acceptably matched. Moreover, when stem cells are provided to the patient, the patient is often near death and needs these cells inserted so that the patient can begin producing their own blood and their own immune system. There is a real chance for death if there is an insufficient number of cells or if the inserted stem cells are not completely sterile. This is because often these patients have been ablated and their immune system completely killed off, so even the slightest infection can be fatal. The end goal is that eventually the new cells inserted will establish a new immune system for the patient. To increase the likelihood of the immediate availability of an acceptable match, a large registry of potential bone marrow donors is maintained and a large cryopreserved inventory of cord blood donor stem and progenitor cells is maintained in a sterile condition to be made available for any patient in need. Blood substitution and blood supply is a permanent strategic and logistical problem of medical services around the world because blood, which is a biological drug, has a limited shelf life and requires special transport and stringent conditions of use. This is especially true with cord blood, and as a result, this source of stem and progenitor cells is typically in a frozen state so they may be collected and stored long before use.
Long term storage of blood cells and other living cells after they have been removed or separated from a donating organism has long been accomplished through freezing. The cryopreservation and recovery of such cells, however, has proven to be quite troublesome. Simple physics dictates that the cells are subjected to relatively harsh conditions during both the freezing and thawing cycles involved in cryopreservation, often resulting in a low survivability rate. The destruction of the cells occurs as the external medium freezes and the cells attempt to maintain equilibrium, thereby losing water and ultimately increasing intracellular solute concentration until intracellular freezing occurs at temperatures below 0 degrees C.
Substantial time and effort has been expended in an effort to maximize the viability of thawed cells, yet there are no known approaches that adequately solve the problem and provide an automatic record of cell survivability. Such efforts have generally focused on the development of cryoprotective agents, the insertion of said cryoprotective agents into the cell solution, and the establishment of optimal cooling and warming rates.
Protection of cells from freezing is achieved by adding so-called cryoprotective agents. Since these cryoprotectants usually cause a significant increase in osmolality, it is nevertheless necessary to have all the procedures monitored, and to have osmotic changes under control to avoid irreversible damage to cellular structures and membranes. Cryoprotection by solute addition of a cryoprotective agent occurs through two mechanisms. The first is intracellular; wherein the amount of ice formed within the cell is reduced throughout the process. The second is extracellular, wherein water flow out of the cell decreases in response to a decreased vapor pressure caused by the formation of ice in the solute surrounding the cells.
Dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant for mononuclear cells, including the hematopoietic and mesenchymal stem and progenitor cells resident in umbilical cord blood, bone marrow, and adipose derived stromal vascular fraction cells. DMSO is an organosulfur compound having the formula (CH3)2SO. When added to cell media, DMSO reduces ice formation and thereby maximizes cell viability during the freezing and thawing process.
However, the introduction of DMSO to these cell solutions is accompanied by a substantial downside, specifically the exothermic release of heat inherent as DMSO and H2O mix. This heat may be of an intensity sufficient to kill the cells if not strictly controlled. Furthermore, DMSO is a powerful solvent that should minimally contact cells directly when in high concentrations. Providing a means of assuring the sterility of the cell solution while adding the DMSO, controlling the rate of introduction of the DMSO solution and assuring its rapid, homogenous distribution within the cell solution is thus both critical and difficult. Moreover, it is very desirable that the cell solution is properly mixed with the DMSO solution by eliminating formation of hot spots and standing waves. Because of these risks, historically a clinician carefully and by syringe pumped or by hand, introduced DMSO solution into a cell solution sample that was sandwiched between ice packs to absorb the exothermic heat. While these methods can be successful for a skilled clinician, they leave too much room for error. Automated record keeping of the mixing process and fluid temperatures is traditionally not present.
Because of the above it is, unfortunately, not uncommon to discover low viability of cells after thawing. When this occurs, it is often not known at which stage in the entire cell processing workflow the failure occurred. There is thus a need to more carefully monitor both the precise mixing of the DMSO and the cell solution and the temperature of the cell solution during and after the introduction of DMSO to that cell solution.
Often it is necessary to provide a patient with multiple dosing from a single donor sample. For this to occur, there is a need to separate the donor cell solution into multiple portions, each in their own hermetically sealed container and each container joined to the other containers to ensure the identity and common controlled freezing rate of each compartment for quality control. Further, while stored at cryogenic temperatures, each hermetically sealed container must also be able to be separated and retrieved from the other joined containers, without piercing the hermetic seal of any of the containers, allowing each container to be thawed separately from the others.
One existing cryopreservation method describes isolating and cryopreserving of human white cells from whole blood. The sequence of operations leading to the separation of red cells, white cells, platelets and plasma is presented in a flow diagram of a bag system for the cryopreservation of leukocytes. Freezing of the white cells is accomplished by the introduction of a combination of hydroxyethyl starch (HES), which functions as both a sedimenting agent and a cryoprotective agent, DMSO. A preferred combination is 4% HES with 5% DMSO. White cell separation is interfaced with collection methods for plasma and platelets so as to conserve all major cell types. However, such a method does not include a means to pump a cryoprotectant solution to the cell solution in a slow, calculated and monitored rate under completely sterile conditions. Moreover, this method does not allow the nutation mixing of the contents of the cell solution and mixing of cell solution with a cryoprotectant solution by eliminating standing waves formed during the mixing processes.
Another existing method describes cryopreservation of peripheral blood lymphocytes. This method comprising the steps of: freezing cells and a cryopreservation medium wherein the cells are freshly isolated lymphocytes, hematopoietic stem cells, lymphocytes which are modified ex vivo, or a combination thereof, wherein the medium does not comprise DMSO or serum, and wherein the arabinogalactan in the medium results in a high post-thaw survival rate for the freshly isolated cells which are modified ex vivo. However, this method does not allow the nutation mixing of the contents of the cell solution, or the mixing of cell solution with a cryoprotectant solution by eliminating standing waves formed during the nutation mixing processes. In addition, this method does not ensure all cells in the cell solution within a container remain in the cell solution and do not attach to the internal surface of the container.
Yet another existing auto-nucleating cryopreservation device includes a tube containing a crystalline cholesterol matrix. The ends of the tube are closed by a membrane that is impermeable to the cholesterol but permeable to liquids contained in a cryopreservation vessel. The auto-nucleating device provides a site for ice nucleation during freezing of the liquid within the vessel. One such cryopreservation vessel is a flexible vial having a closed port at one end adapted to be pierced by a needle to withdraw the liquid within, and an opposite end that is initially open to receive the liquid. Another vessel includes an adaptor mounted to liquid container with a tubular branch closed by a needle septum, and the other tubular branch provided with a barbed fitting for engaging a flexible tube that terminates in the needle septum. In another embodiment, the vessel includes an inlet and vent branch at the top of the container and an outlet septum at a bottom opening. However, this device does not allow nutation mixing of the contents of the cell solution and the mixing of the cell solution with a cryoprotectant solution under sterile conditions by eliminating standing waves formed during the nutation mixing process. Moreover, the device cannot regulate, maintain, and record temperature of the mixture of the cell solution and the cryoprotectant solution before, during and after the cryoprotectant solution insertion.
Various other cryopreservation devices and methods currently available do not include a means to regulate temperature of the cell solution within a mixing container by conduction due to direct heat transfer. Finally, U.S. Pat. No. 8,747,289 B2 (application Ser. No. 13/634520, filed by the Applicant on Mar. 17, 2011 and claiming priority to U.S. Provisional app. 61/315109 filed Mar. 18, 2010 and U.S. Provisional app. 61/436964 filed Jan. 27, 2011) describes an apparatus and method for purifying and harvesting certain cell populations in blood or bone marrow by depleting at least one of red blood cells, granulocytes, or platelets from a sample comprising blood, bone marrow, or stromal vascular fraction cells separated from adipose tissue. The apparatus provides the capture of and means for increasing the concentration of certain cell types. The apparatus comprises a sterile, single use rigid, self-supporting cartridge within which the automated depletion, purification and harvesting of target cell populations occurs and all components may be distributed. FIG. 4 illustrates an example of a prior art rigid disposable cartridge within which the certain cell types are concentrated. The rigid disposable cartridge 150 is cylindrical, single-use, and constructed preferably of hard plastic, and more preferably optically clear polycarbonate. The rigid disposable cartridge 150 includes an output tubing 152 that can be connected to at least one tubing to provide for a transfer of materials from the rigid disposable cartridge 150. A top portion of the disposable cartridge includes a 0.2-micron filter 154 to provide passage for displaced air from within the funnel when blood or bone marrow is introduced into the funnel. The control module 156 in which the disposable cartridge 150 is seated is a battery-operated, electro-mechanical device with optical and gravitational sensing. The preferred embodiment also comprises a membrane switch 158, a seven segment digital read out 160 and three light emitting diodes 162 to inform and assist the user. A universal battery indicator 164 alerts the user to the charge condition of the battery. Shown in the center is an on-off switch 166 for the control module and a Light Emitting Diode (LED) 162, and on the right is the seven segment digital read out 160 and the LED 162 that indicates whether the cell harvest run was performed as designed and, if not, which error in operation may have occurred. Although the apparatus and related method of use are highly useful, such a method does not include a means that cryopreserves the collected cells.
There is thus a need for a simple, universal processing set that provides an optimum environment for cryoprotecting a clinically relevant population of stem and progenitor cells, while maintaining the sterility and viability of the cells and facilitating their long-term storage at cryogenic temperatures. Such an apparatus and method would include individual sealed containers, each containing a portion of the original volume of collected bone marrow or cord blood containing essentially all the nucleated cells, and be individually cryoprotected and frozen in a manner that assures a high degree of post-thaw cell viability. Such an apparatus and method would include a means to pump a cryoprotectant solution to the cell solution in a slow, calculated and monitored rate under completely sterile conditions. Such an apparatus would preferably include a container that is configured to allow the nutation mixing of the contents of the cell solution and mixing of the cell solution with a cryoprotectant solution by eliminating standing waves formed during the mixing processes. Such an apparatus and method would ensure all cells resident in a cell solution within a container are contained in the cell solution and do not adhere to the internal surface of the container. Such a system would include a means to regulate the temperature of the cell solution within a mixing container by conduction due to direct heat transfer. This apparatus would be configured to communicate with a storage server so as to transmit data gathered during the method for cryopreservation of biological materials. Furthermore, such an apparatus and method would include the container that optimizes the rate of dissemination of DMSO so that the DMSO is quickly and homogenously diffused, consequently reducing incidents of cell death. Further, this apparatus and method would provide a workstation in which the temperature of the mixture of the harvested cell solution and the cryoprotectant solution before, during and after cryoprotectant solution insertion is regulated, maintained and recorded.